The large conductance calcium-activated potassium (BK) channels are major ionic determinants in mediating vasorelaxation and are the target of endothelium-derived hyperpolarizing factors (EDHFs). We found that regulation of BK channels by EDHFs is abnormal in diabetic animals and the intrinsic properties of BK channels are altered in diabetic coronary arteries. The BK channels in coronary arterial smooth muscle cells from diabetic animals have reduced sensitivity to calcium- and voltage-dependent activation. We have demonstrated that the mechanism whereby vascular BK channel regulation is altered in Type 1 and Type II diabetes involves hyperglycemia-induced oxidative stress, where the cysteine residues in BK channels are targets of redox modulation. The C911 residue of the BK channel pore subunit is particularly sensitive to modulation by hyperglycemia and by hydrogen peroxide, as the C911A mutation is insensitive to the effects of hydrogen peroxide and to high glucose. The goal of this project is to further delineate the molecular mechanisms through which reactive oxygen species (ROS) modulate BK channels in diabetes. The hypotheses to be tested are: 1) Abnormal vascular BK channel function in diabetes is due to redox modulation of specific cysteine residues by ROS. 2) BK channel modified by ROS is a substrate for ubiquitination and proteasomal degradation. Three specific aims are proposed. Aim 1 will examine the mechanism of BK channel modulation by diabetes-induced oxidative stress. Aim 2 will examine whether prevention of oxidative modulation of specific cysteine residues would maintain the integrity of BK channel function in diabetes. Aim 3 will determine whether redox-modulated BK channels are targets for ubiquitination and proteasomal degradation. These studies will be performed using in vitro and in vivo models of diabetes. Whole-cell and single channel patch clamp techniques, antioxidant enzyme in vitro gene transfer, and specific transgenic mice will be employed to determine the effects of diabetes-induced ROS production on BK channel function and degradation. The results of this project may provide important novel insight into the electrophysiological and molecular mechanisms of altered BK channel function that may contribute to both endothelium-dependent and -independent vascular dysfunction in diabetes. PUBLIC HEALTH RELEVANCE: Diabetes has become a disease of epidemic proportions and cardiovascular diseases are the most important causes of morbidity and mortality in diabetic patients. BK channels are proteins that regulate vessel tone and are critical determinants of blood flow in vital organs. We have found that the function of BK channels is abnormal in diabetic coronary arteries. The goal of this project is to delineate the molecular mechanisms underlying these changes. We hypothesize that BK channels in diabetes are abnormal as a result of enhanced oxidative stress and the fate of "oxidized" BK channel is accelerated degradation. The results of this project may provide important novel insights into the mechanisms through which diabetes leads to abnormal function in the cardiovascular system.